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Colombia Médica Vol. 41 Nº 2, 2010 (Abril-Junio)

Determination of the prevalence of hemoglobin S, C, D, and G inneonates from Buenaventura, Colombia*

MATILDE DE BERNAL, MD1, ANDRÉS COLLAZOS, MD2,RUBÉN DARÍO BONILLA, BIOL3, E DNA PATRICIA TASCÓN, B IOL4

SUMMARY

Introduction: Inherited hemoglobinopathies are common among African Blacks. In Buenaventura, a city on Colombia’sPacific coast, where 92% of the population is Afro-Colombian, there are few published attempts to identify these disorders.Affected individuals require more health care due to higher morbidity and mortality. Early identification of these newbornsfollowed by comprehensive care is important to reduce co-morbidities.

Objective: To study newborns and establish the numbers at risk with a bloodspot screening method. This informationwill demonstrate to Public Health Authorities the need to provide care for this population.

Methods: A cross-sectional descriptive study of a sample of 399 newborns (95% CI) where there is an expected prevalenceof 10% of abnormal hemoglobins. Mothers in at least the 36th week of gestation, living in the urban area of Buenaventura,were used. Umbilical cord blood was drawn and specimens fixed on filter paper and stored at 4°C. Isoelectric focusingelectrophoresis assays were used to separate the hemoglobins. The results were reported according to the identifiedhemoglobin as F, A, S, C, D, and G.

Results: We processed 399 samples, 353 (88.5%) were normal (hemoglobin FA), 23 (5.8%) were heterozygous forhemoglobin C (FAC), 19 (4.8%) were heterozygous for hemoglobin S (FAS), 2 (0.5%) were heterozygous for hemoglobinG (FAG), 1 was heterozygous for hemoglobin D (FAD) and 1 was heterozygous combined S and C (FSC).

Conclusion: Hemoglobins S, C, D, and G are common among infants born in Buenaventura. Hemoglobin C occurredmore frequently than in other reported studies. This study suggests that both detection and a follow-up program arerequired in areas with a high density of Afro-Colombian population.

Keywords: Prevalence; Newborn; Hemoglobinopathies; Hemoglobin S; Hemoglobin C; Anemia;Sickle cell; Electrophoresis, iso electric point; Colombia.

Determinación de la prevalencia de la hemoglobina S, C, D y G en recién nacidos de Buenaventura, Colombia

RESUMEN

Introducción: Las hemoglobinopatías son entidades muy frecuentes en el África negra. En Buenaventura, ciudad de lacosta pacífica colombiana con una población 92% afrocolombiana, hay pocas publicaciones identificando esta patología. Laspoblaciones afectadas requieren cuidados especiales por su alta mortalidad y morbilidad. La identificación temprana desdeel periodo neonatal y los programas de cuidado integral se imponen en poblaciones de alta prevalencia.

Objetivo: Determinar la prevalencia en Buenaventura de hemoglobinas S, C, D y G con un modelo de tamizaje neonatal.La información colectada servirá para sustentar recomendaciones a las autoridades de salud pública.

Metodología: Se realizó un estudio descriptivo de corte transversal en 399 neonatos para una prevalencia esperada del

* Resources from the Endocrinology Laboratory at Universidad del Valle and co-financing through resources from aninternal call for research projects at Universidad del Valle.

1. Full Professor, Department of Internal Medicine, Endocrinology Laboratory, Universidad del Valle, Cali, Colombia.e-mail: [email protected]

2. Assistant Professor, Universidad del Valle, Department of Internal Medicine, Universidad del Valle, Cali, Colombia.e-mail: [email protected]

3. Biologist, Department of Internal Medicine, Endocrinology Laboratory, Universidad del Valle, Cali, Colombia.e-mail: [email protected]

4. Biologist, Universidad del Valle, Cali, Colombia. e-mail: [email protected] for publication December 7, 2009 Accepted for publication January 12, 2010

© 2010 Universidad del Valle, Facultad de Salud Colomb Med. 2010; 41: 141-7

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10% (IC 95%), con madres residentes del área urbana deBuenaventura y 36 semanas o más de gestación. Se tomómuestra de sangre de cordón umbilical fijada en papel filtro yalmacenada a 4°C. La técnica de electroforesis de puntoisoeléctrico sirvió para la separación de las hemoglobinas.Los resultados se informaron de acuerdo a las hemoglobinasidentificadas F, A, S, C, D y G.

Resultados: Se procesaron 399 muestras de cordón. El88.5% (353) de las muestras fueron normales (hemoglobinaFA), 23 (5.8%) fueron heterocigotos para hemoglobina C(FAC), 19 (4.8%) fueron heterocigotos para hemoglobina S(FAS), 2 (0.5%) fueron heterocigotos para hemoglobina G(FAG), 1 fue heterocigoto para hemoglobina D (FAD) y 1 fueheterocigoto combinado S y C (FSC).

Conclusión: Hemoglobinas S, C, D y G son frecuentesentre los neonatos de Buenaventura. La hemoglobina C fuemás frecuente que en estudios previos, probablemente porsesgos en la selección de la muestra de dichos estudios. Sesugiere a las autoridades de salud pública la ampliación delprograma de tamizaje de enfermedades congénitas como lashemoglobinopatías.

Palabras clave: Prevalencia; Recién nacido;Hemoglobina S; Hemoglobina C; Anemia de células

falciformes; Electroforesis de punto isoeléctrico;Colombia; Hemoglobinopatías.

Neonatal screening for abnormal hemoglobin (Hb)permits establishing the presence of hemoglobinopathiesin the population. Sickle-cell disease is one of these, withthe disorder being most frequent in countries with obligatoryneonatal screening1,2. It is produced by the specific mutationof the beta-globin gene3, resulting from the change ofglutamic acid for valine in the 6th position, of autosomalrecessive inheritance. The most common genotypes are:homozygous hemoglobin S (HbS), combined heterozy-gous SC (HbSC), and Sβ+ thalassemia and Sβ0 thalassemia;and, less frequently, it is due to the combination of hemoglobinS with other variants (for example, hemoglobin D, G).Sickle-cell disease increases morbidity and mortalityduring the first years of life4, being greater in homozygousSS individuals in comparison to heterozygous SC.Hemoglobin C is associated to less symptoms and offersprotection against severe malaria5.

The high prevalence of sickle-cell anemia in apopulation with African ancestry was shown in theanalysis of records from Texas from 1992 to 1998, from2’292,698 live births and 94% specimens collected,revealing total prevalence per ethnic groups per every

10,000 live births of 29.91 Afro-Americans, 0.11 White,0.29 Hispanic, and 2.47 others6. The studies conductedin the city of Buenaventura and in other towns in theColombian Pacific region are: from a reference centerfor malaria infection in individuals older than one year ofage that showed a prevalence of 14.9% of abnormalhemoglobins (AS of 7%, AC of 6.2%, and AD of1.7%)7; from the Hematology service at Universidaddel Valle in individuals referred because of havingsamples suspicious of sickle-cell anemia8, and fromSalahonda (close to Tumaco in Nariño, Colombia) withsickle-cell trait in 10% of the population and clinicalrelevant hemoglobinopathies by 1%9.

A preliminary report published in 2004 of neonatalscreening begun in Cali (Valle del Cauca, Colombia, withan Afro-Colombian population of 26.51%) as of 2003 of6500 neonates, revealed 146 cases of HbS carriers (2.4%),90 cases heterozygous HbC (1.4%), 2 heterozygous HbD(0.02%), and 2 homozygous HbC, without cases ofhomozygous S10; however, there is no definition ofinclusion criteria (especially of gestational age) and ofexclusion criteria (transfusions prior to taking the sample).There is no knowledge of the prevalence of the types ofabnormal hemoglobins (S, C, D, and G) in newborns in thecity of Buenaventura (Valle del Cauca, Colombia). Thiscity, located in the Pacific region, has a populationprojected for 2005 of 278,960 inhabitants (86% living in theurban zone)11. The proportion of Afro-Colombian populationis of 92%. Previous studies of cost-effectiveness analysisfavor neonatal detection aimed at, that is, only newbornsfrom some ethnic groups12,13 when the individuals at riskare few among the population; however, universal detectionidentifies more newborns with the disease and preventsmore deaths14,15.

Early detection of homozygous S newborns permitsthe application of broadly recognized integral programsin health services, vaccines, monitoring, and educationfor children and their parents2, diminishing the incidenceof infection and associated mortailty16. Parent educationon disease and above all the symptoms (e.g., splenicsequestration) permits opportune attention of severecases. The possibility of curing via allogenic stem celltransplants informed in the literature, although stillpromissory, assigns more relevance to detection17.

The objective of this study was to determine theprevalence of hemoglobins S, C, D, and G in newbornsfrom a high-risk zone like the city of Buenaventura.

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METHODS

A descriptive cross-sectional study was conducted.Universal screening was employed15.

To estimate the sample size, we used the informationon the projected population for the city of Buenaventurafor 2005, because the data for the 2005 census andprojections for subsequent years had not beenpublished18. Said projection reports 278,960 inhabitants(86% located in the urban zone)11. To estimate thesample size, the StatCalc 6 th version program by EpiInfowas used based on the following considerations:estimated prevalence of hemoglobinopathies (S, C, D,G) equal to 10%9, margin of error of 3%, and confidencelevel of 95%. The representative minimum sample sizewas of 384 newborns.

The samples were taken from newborns at threehospitalization centers in the city: Departmental Hospi-tal of Buenaventura – complexity level 2, Luis Ablanquede la Plata Hospital – complexity level 1, and theBuenaventura Clinic – complexity level 2 (these are thehospitalization centers in Buenaventura authorized forchildbirth attention by the Valle del Cauca DepartmentalSecretary of Public Health), these were taken afterinformed signed consent by legal guardians.

Inclusion criteria. Gestational age of 36 or moreweeks (corroborated by obstetric sonogram, given thatmore premature newborns do not express in adequateproportion the hemoglobins of the adult stage19), domiciledin the urban area of Buenaventura, born between 01February 2007 and 01 February 2008. On an adjoiningcard, the following data were registered: mother’s name(or legal guardian), mother’s age (in years), telephonenumber, home address, gestational age, date of birth,sex (female or male), and weight of the newborn.Exclusion criteria: newborns with red blood celltransfusion prior to taking the sample.

Taking of blood sample and storage. Using asyringe, 1 ml of blood was drawn from the umbilical cordplacental end in loop form after its ligation (or from theheel, in case of not having been obtained from the firstsite or due to bad previous sample taking) and a dropwas fixed on each circle of the filter paper (a total of 4drops), and stored at 4ºC.

Analytical methods. The isoelectric focusingelectrophoresis (IFE) technique was selected becauseof the low costs of equipment and samples, high sensitivity

(100%), and specificity (100%) compared to methodslike electrophoresis in cellulose agar and high-perfor-mance liquid chromatography20 with low intra- andinter-assay error21, permitting reproduction of the results.With IFE, each hemoglobin has a defined isoelectricpoint (pI) (Table 1). To develop the methodology, weused the Wallac RESOLVE® Neonatal Hemoglobintest kit (PerkinElmer, 940 Winter Street, Waltham,Massachusetts 02451, USA)21,22.

The results of the hemoglobins were reported inqualitative order according to the percentage of hemo-globins present like: FA (normal), FS (homozygous HbS), FAS (carrier Hb S), FC (homozygous Hb C), FAC(carrier Hb C), FSC (mixed S and C heterozygous),FAD (carrier Hb D), and FAG (carrier Hg G) (Graph 1).

The statistical analysis was conducted based onpercentage distributions, prevalence, and confidenceintervals of 95% - according to the exact binomialmethod using the Epi Info Software for Windows,version 3.3.2.

Ethical considerations. The study was approvedby the Institutional Committee on Human Ethics Reviewof the Faculty of Health at Universidad del Valle. Thesample was taken from each newborn after informedsigned consent from one of the parents or legal guardian.

RESULTS

In total, 399 samples were collected from newbornsat three Institutions: Departmental Hospital of Buena-ventura 252 (63.2%), Luis Ablanque de Plata Hospital113 (28.3%), and Buenaventura Clinic 34 (8.5%). Some98.7% of the samples were taken from the umbilicalcord; the rest were collected from the heel.

Table 1Isoelectric points (pI) of the hemoglobin variants.

Resolve Isoscan (Perkin–Elmer) system

Hemoglobin pI

A 6.90F 7.00G, Philadelphia 7.22D 7.25S 7.28C 7.65

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A total of 50.6% of the newborns were females. Theaverage gestational age was 39.3 ± 1.1 weeks, withaverage weight at birth of 3114 ± 505 g. The prevalenceof newborns with at least one abnormal hemoglobin wasof 11.7%, with 8.6%-15.1% having a confidence interval

of 95% (Table 2, Graph 2, Graph 3, Graph 4). Nosignificant statistical differences were found regardinggender or the gestational age of the newborns withrespect to the hemoglobin pattern.

Graph 1. Hemoglobin isoelectric focusing electrophoresis

Pattern: reference pattern. FAS: heterozygous hemoglobin S. FAC: heterozygous hemoglobin C. FA: normal. Sample processed in theEndocrine Laboratory at Universidad del Valle.

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Sample processed in the Endocrine Laboratory at Universidad del Valle

Graph 3. Identificationof the heterozygoushemoglobin D via iso-electric focusing elec-trophoresis

Graph 2. Identification ofthe heterozygous hemo-globin G via isoelectricfocusing electrophoresis

Graph 4. Identification of combined heterozygousSC via isoelectric focusing electrophoresis

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DISCUSSION

Although the total percentage of abnormal hemo-globins in this study is similar to prior studies7-9, we notea greater proportion of newborns with hemoglobin C, incomparison with previous studies in the zone. HemoglobinC is more frequent in Western Africa23 and it is associatedto minor symptoms related to malaria infection5,24.Homozygous C has lower complications related tosickle-cell anemia when compared to homozygous S.They may even be asymptomatic into adulthood, althoughthe retinopathy related to sickle-cell anemia is morefrequent with hemoglobin C25, requiring periodic ophthal-mologist control of these individuals.

The previously mentioned studies conducted in adultsand children older than one year of age could under-estimate the prevalence in the population when notincluding the children who perished because of sickle-cell disease before reaching one year of life, and notincluding individuals with hemoglobin C that may nothave attended the center of reference for malariainfection (because of manifesting few or no symptoms).

Only one newborn with combined heterozygous SCwas found, warranting a periodic monitoring. In theseindividuals, it was not establish the effectiveness of theantibiotic prophylaxis to diminish mortality due topneumococcus, which has indeed benefited homozygousSS individuals. The current study found no homozygousS or C, given that the design was not made for individualswith the disease of sickle-cell anemia; this requires alarger sample size. When the screening detects a newbornwith hemoglobins FS or FC, the test must be confirmed

before two months of age to discard the co-existence ofbeta+ thalassemia26. In 2007, 4546 babies were born inthe urban zone of Buenaventura27. The study sample of399 newborns represents 8.7% (399/4546) of the livebirths for 2007, hence, applying for this period.

The high prevalence of hemoglobin alterations in ahigh-risk population, like Buenaventura, suggests theneed for an universal neonatal detection program inthese types of zones. This should be done for thepurpose of early detection of homozygous S or Cindividuals, and opportunely initiate preventive andeducational measures tending to diminishing morbidityand mortality related to sickle-cell anemia.

Conflict of interest. None of the authors has conflictsof interest related to this study.

ACKNOWLEDGMENTS

We thank the directors and personnel from theneonatal wards at the Departmental Hospital in Buena-ventura, the Luis Ablanque de la Plata Hospital, and theBuenaventura Clinic for their collaboration. We alsothank John Jairo Lozano and María del Pilar Silva R,professionals at the Endocrine and MetabolismLaboratory in the Faculty of Health at Universidad delValle for their collaboration in administrative and budgetmatters and in the transcription of documents.

REFERENCES

1. Weatherall D, Clegg J. Inherited haemoglobin disorders: anincreasing global health problem. Bull WHO. 2001; 79: 704-12.

2. American Academy of Pediatrics, Section on Hematology/Oncology, Committee on Genetics. Health supervision forchildren with Sickle Cell Disease. Pediatrics. 2002; 109: 526-35.

3. Steinberg M. Management of Sickle Cell Disease. N Engl J Med.1999; 340: 1021-30.

4. Platt O, Brambilla D, Rosse W, Milner P, Castro O, SteinbergM, et al. Mortality in sickle cell disease. Life expectancy andrisk factors for early death. N Engl J Med. 1994; 330: 1639-44.

5. Agarwal A, Guindo A, Cissoko Y, Taylor J, Coulibaly D, KonéA, et al. Hemoglobin C associated with protection from severemalaria in the Dogon of Mali, a West African population witha low prevalence of hemoglobin S. Blood. 2000; 96: 2358-63.

6. Strahan, J, Canfield M, Drummond-Borg L, Neill S. Ethnic andgender patterns for the five congenital disorders in Texas from1992 through 1998. Tex Med. 2002; 98: 80-6.

7. Moyano M, Méndez F. Defectos eritrocíticos y densidad dela parasitemia en pacientes con malaria por Plasmodiumfalciparum en Buenaventura, Colombia. Rev Panam Salud

Table 2Prevalence (%) of hemoglobins in newborns in

Buenaventura, Colombia

Hemoglobin pattern Number Prevalence %

FA 353 88.5FAC 23 5.8FAS 19 4.8FAG 2 0.5FAD 1 0.3FSC 1 0.3Total 399 100.0

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Publica. 2005; 18: 25-32.8. Pereira F, Sáenz I. Hemoglobinopatías en niños. Colomb Med.

1996; 27: 146-9.9. Muñoz N, Pereira F, Sáenz I. Hemoglobinas anormales en

Salahonda (Tumaco). Acta Pediatr Colomb. 1994; 1: 7-10.10. Satizábal J, Neuta P, Muñoz J, Somoyar P. Incidencia de

hemoglobinopatías en neonatos de Cali. Salud Uninorte. 2004;18: 71-2.

11. DANE. Proyecciones de población, por área, según munici-pios 1995-2005. Bogotá, DC: DANE.

12. Panepinto J, Magid D, Rewers M, Lane PA. Universal versustargeted screening for sickle cell disease: a cost effectivenessanalysis. J Pediatr. 2000; 136: 201-8.

13. Tsevat, J, Wong, J, Pauker, S, Steinberg, M. Neonatal screeningfor sickle cell disease: A cost-effectiveness analysis. J Pediatr.1991; 118: 546-54.

14. Henthorn, JS, Almeida, AM, Davies, SC. Neonatal screeningfor sickle cell disorders. Br J Haematol. 2004; 124: 259.

15. Consensus Development Panel. National Institutes of Health.Newborn screening for SCD and other hemoglobinopathies.JAMA. 1987; 258: 1205-9.

16. Gaston M, Verter J, Woods G, Pegelow C, Kelleher J, PresburyG, et al. Prophylaxis with oral penicillin in children with sicklecell anemia. A randomized trial. N Engl J Med. 1986; 314: 1593-9.

17. Bernaudin F, Socie G, Kuentz M, Chevret S, Duval M,Bertrand Y, et al. Long-term results of related myeloablativestem-cell transplantation to cure sickle cell disease. Blood.2007; 110: 2749-56.

18. Departamento Administrativo Nacional de Estadística. Sínte-sis del proceso de cierre del Censo General 2005. Bogotá:DANE; 2008.

19. Telen M. The mature erythrocyte. En: Greer JP, Foerster J,Rodgers, GM, Paraskevas F, Glader B, Arber DA, et al.

Wintrobe’s clinical Hhematology. 12th ed. Philadelphia:Lippincott Williams & Wilkins; 2008. p. 126-55.

20. Sickle Cell Disease Guideline Panel. Sickle Cell Disease:screening, diagnosis, management, and counseling in newbornsand infants. Clinical practice guideline N° 6. AHCRP Pub. N°93-0562. Rockville: Agency for Health Care Policy and Research,Public Health Service, U.S. Department of Health and HumanServices; 1993.

21. Hempe J, Craver R. Quantification of hemoglobin variants bycapillary isoelectric focusing. Clin Chem. 1994; 40: 2288-05.

22. Basset P, Beuzard Y, Garel M, Rosa J. Isoelectric focusing ofhuman hemoglobin: its application to screening, to thecharacterization of 70 variants, and to the study of modifiedfractions of normal hemoglobins. Blood. 1978; 51: 971-82.

23. Devoucoux R, Hurpin C, Baudon D, Molez J, Roux J, Guilloud-Bataille M, et al. Population genetics of abnormal haemoglobinsin Burkina Faso, west Africa. Ann Hum Biol. 1991; 18: 295-302.

24. Rihet P, Flori L, Tall F, Traoré A, Fumoux F. Hemoglobin Cis associated with reduced Plasmodium falciparum parasitemiaand low risk of mild malaria attack. Hum Mol Genet. 2004; 13:1-6.

25. Condon P, Gray R, Serjeant G. Ocular findings in children withsickle cell haemoglobin C disease in Jamaica. Br J Ophthalmol.1974; 58: 644-9.

26. Strickland D, Ware R, Kinney T. Pitfalls in newbornhemoglobinopathy screening: Failure to detect ß+-thalassemia.J Pediatr. 1995; 127: 304-8.

27. Departamento Administrativo Nacional de Estadística. Cua-dro 7. Nacidos vivos por sitio del parto, según departamento,municipio y área de ocurrencia. Nacimientos 2007. URLdisponible en: http://www.dane.gov.co/files/investigaciones/poblacion/nacimientos/nac_07/Cuadro7.xls

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